1. Field of the Invention
The present invention relates to binary probes for detecting nucleic acids.
2. Description of the Related Art Invention
Single nucleotide polymorphisms (SNPs) are the most abundant forms of genetic variations in the human genome. Large-scale sequence analysis is needed for a population-based genetic risk assessment and diagnostic tests once a mutation has been identified. However, most of the methods for SNP screening require enzymatic manipulations such as endonuclease digestion, ligation or primer extension, and often separation of the resultant products.1 These labor intensive and time consuming procedures are some of the biggest impediments to moving SNP typing techniques to point-of-care settings, which require straightforward, inexpensive, and disposable detection formats. Towards fulfilling of these requirements a probe for visual SNP detection was developed in this study.
Binary probes for fluorimetric analysis of single nucleotide substitutions were developed earlier.2 The probes demonstrate improved selectivity in comparison with conventional hybridization-based approaches, since the two parts of the probes form relatively short (7-10 nucleotide) duplexes with target sequences. These short hybrids are extremely sensitive to single nucleotide substitutions at room temperature and generate high fluorescent signal only in the presence of the fully complementary targets. Binary probes do not require precise temperature control for SNP typing.2d,e However, a fluorimeter is required for signal registration.
Earlier, gold nanoparticle (GNP)—based approaches were suggested for SNP typing with a colorimetric/optical outcome.7 These methods involve attaching of non-complementary DNA oligonucleotides capped with thiol groups to the surface of two batches of 13-nm GNPs. When DNA, which is complementary to the two engrafted sequences, is added to the solution, a polymer network is formed. This condensed network brought the conjugated GNPs to self-assemble into aggregates with a concomitant red-to-purple color change. Alternatively, a non-crosslinking DNA-GNP aggregation method takes advantage of blunt end stacking interactions of the DNA double helixes.8a All these approaches demand conjugation of DNA probes with colloid gold. In addition, some of the techniques require precise temperature control for allele discrimination. These procedures complicate both the probe preparation and the assay itself. Unlike gold nanoparticle-based approaches, a binary DNA peroxidase probe requires neither postsynthetic modification of the probe oligonucleotides, nor precise temperature control for SNP typing.
There is still a need for a DNA probe that retains sensitivity while permitting visual detection of a DNA analyte.